This application is submitted in response to RFA HL-02-018. Mesenchymal stem cells (MSC) can be expanded and induced to differentiate into a variety of cell types of mesenchymal origin thus providing the potential to replace or restore tissues damaged by disease. In addition, the use of MSC with inserted genes could provide a means for integration of healthy cells into host tissue with gene products that can restore or enhance organ function. The goal of this proposal is to explore the use of rhesus monkey MSC (rhMSC) derived from different aged donors for fetal transplantation and gene delivery. In Specific Aim 1 we will evaluate the growth characteristics, cell cycle status, and phenotypic markers of rhMSC subpopulations when derived from fetal, newborn, and adult donors. Oligonucleotide microrarray-based gene expression analysis will also be performed, and quantitative RT-PCR will be used to confirm the transcript levels of groups of genes that may serve as cell markers. We will also assess whether rhMSC transduction with lentiviral vectors alters critical biochemical pathways that could potentially impact on the engraftment and differentiation capabilities of these cells in vivo. Specific Aim 2 will focus on fetal transplant of male donor-derived rhMSC characterized in Specific Aim 1. Engraftment and differentiation will be evaluated using Y chromosome-based assays. The results of these studies will be compared to transplant with transduced rhMSC from these same donors using SIN lentiviral vectors. Flow cytometric, immunohistochemical, RT-PCR, and morphologic techniques including laser capture microdissection will be used to evaluate donor cell engraftment and differentiation in vivo. Cell concentration and gestational timing of the transplant will also be explored, and the potential postnatal ramifications of prenatal intervention evaluated as the animals mature. The outcome of these studies will be correlated with gene expression profiles obtained in Specific Aim 1. By addressing basic questions on rhMSC biology in vitro, and exploring fetal delivery of defined cell populations in vivo, we will assess essential questions in an established monkey model system that will provide the basis for future clinical application, and address potential new treatment strategies for human disease.